Different effects of lobeline on neuronal and muscle nicotinic receptors.

Lobeline is a plant alkaloid known to interact with cholinergic system. The effect of lobeline on neuronal α3ß4 receptors expressed in COS cells and muscle embryonic αßγδ receptors naturally expressed in TE671 cells was studied using a patch-clamp technique. Our results show that lobeline inhibited responses to acetylcholine in human embryonic muscle nicotinic receptor in a pseudo-noncompetitive manner. The responses of rat neuronal α3ß4 receptors to a low concentration of acetylcholine were potentiated by a mixed occupation mechanism that corresponds to "competitive potentiation". This potentiation turned into voltage-dependent inhibition for α3ß4 receptors was activated by a high concentration of acetylcholine.

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats.

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni-quantal end-plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3ß4 and αßεδ acetylcholine receptors in COS cells.

The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3ß4 (ganglionic type) and αßεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3ß4 or αßεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors.

Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm.

We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 µM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca(2+) channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G(q/11) and PLC. ATP thus regulates the neuromuscular synapse by two different pathways.

Dual effect of lobeline on α4ß2 rat neuronal nicotinic receptors.

The effect of lobeline on rat α4ß2 nicotinic receptors expressed in COS cells was studied using the patch-clamp technique. Currents were recorded in whole-cell mode 2-4 days after cell transfection by plasmids coding the α4ß2 combination of receptor subunits. In cells sensitive to acetylcholine, the application of lobeline evoked minor responses (up to 2% of maximal acetylcholine response). When acetylcholine was applied to the background of an already running application of lobeline, acetylcholine responses were inhibited in a concentration- and time dependent manner. However, when lobeline was applied simultaneously with acetylcholine without any prepulse or during an already running application of acetylcholine, the acetylcholine responses were potentiated up to 300-600% of that of the control. The site of lobeline action overlaps with the cholinergic site, as was proven by the partially protective effect of (+)-tubocurarine. Thus, lobeline can apparently desensitize receptors when applied alone (inhibition) whereas its binding to a second agonist site with the first one already occupied by acetylcholine leads to channel opening (potentiation).

Different sensitivities of rat skeletal muscles and brain to novel anti-cholinesterase agents, alkylammonium derivatives of 6-methyluracil (ADEMS).

BACKGROUND AND PURPOSE: The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6-methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain. EXPERIMENTAL APPROACH: Increased amplitude and prolonged decay time of miniature endplate currents were used to assess anti-cholinesterase activity in muscles. In hippocampal slices, induction of synchronous network activity was used to follow cholinesterase inhibition. The inhibitor sensitivities of purified AChE from the EDL and brain were also estimated. KEY RESULTS: The intermuscular difference in sensitivity to ADEMS is partly explained caused by a higher level of mRNA and activity of 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547)-resistant BuChE in the diaphragm. Moreover, diaphragm AChE was more than 20 times less sensitive to C-547 than that from the EDL. Sensitivity of the EDL to C-547 dramatically decreased after treadmill exercises that increased the amount of PRiMA AChE(G4), but not ColQ AChE(A12) molecular forms. The A12 form present in muscles appeared more sensitive to C-547. The main form of AChE in brain, PRiMA AChE(G4), was apparently less sensitive because brain cholinesterase activity was almost three orders of magnitude more resistant to C-547 than that of the EDL. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools.

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles.

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 µM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

Potassium-selective microelectrode revealed difference in threshold potassium concentration for cortical spreading depression in female and male rat brain.

It has been found with potassium-selective microelectrodes that the threshold extracellular concentration of potassium [K(+)](e) for eliciting fast spreading depression from the site of administration 1.5mm away is 80% lower in the rat female cortex (8.0 ± 0.6 mM) than in the male cortex (14.4 ± 0.4mM). The rate of the first slow phase of [K(+)](e) rise before reaching the threshold and speed of SD propagation were similar for both sexes as well as the rise of the second active fast phase of the [K(+)](e) increase. Lower [K(+)](e) threshold in females can facilitate the rapid K(+) release from the nerve tissue cells that may cause a cerebrovascular vasodilatation and the attack of migraine pain.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers.

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca(2+), and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129-132, 2005; Bukharaeva et al., J Neurochem 100:939-949, 2007).

Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors.

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism.

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.

Physostigmine modulation of acetylcholine currents in COS cells transfected with mouse muscle nicotinic receptor.

Physostigmine (Phy), a reversible inhibitor of acetylcholine (ACh) esterase (AChE), may also act as a low potency agonist and a modulator of the nicotinic receptor. The actions of Phy on mouse muscle nicotinic receptors in the COS-7 cell line were studied by the patch-clamp technique. Currents were recorded in the whole-cell mode 3-7 days after cell transfection by plasmids coding alphabetagammadelta combination of receptor subunits. The application of ACh to cells clamped at -10 mV produced inward currents which displayed desensitization. The application of Phy in concentrations up to 1 x 10(-3) M did not give reliable specific whole-cell membrane responses. The application of Phy in concentrations of 10(-6)-10(-4) M together with ACh modulated the amplitude; accelerated desensitization of currents induced by ACh and increased the final extent of desensitization in a concentration-dependent manner. This finding is in contrast to the suppression and slowing down of desensitization by Phy and 1-methyl-galanthamine observed in Torpedo receptors.

[The effect of atropine and diazepam on mammalian neuromuscular junction--a model of their protective action against anticholinesterase-based war and agriculture poisons]. / Pusobeni atropinu a diazepamu na savci nervosvalové ploténce--model jejich ochranného pfusobení pri otrave bojovými a zemedelskymi anticholinesterázami.

The effect of atropine and diazepam on the mammalian neuromuscular junction. Nicotinolytic effect of atropine on the neuromuscular junction is discussed as a main mechanism of the beneficial effect of this drug during war and agriculture poisoning by anticholinesterases. Atropine is beneficial as it reduces the amplitude of intracellularly recorded endplate potentials and, first of all, causes a marked shortening of their time course (Beránek, Vyskocil 1968, Magazanik, Vyskocil 1969). Diazepam effectively blocks trains of action potentials in individual rat diaphragm muscle fibers, apparently by elevating the chloride permeability. It is suggested that similar increase in Cl- permeability may occur in brain excitable structures and can counteract the anticholinesterase-induced prolongation of ACh-depolarization that evokes repetitive firing.

Allosteric modulation of the nicotinic acetylcholine receptor by physostigmine.

The action of physostigmine on mouse muscle nicotinic acetylcholine receptor expressed in the COS-7 cell line was studied by the patch-clamp technique. Physostigmine accelerated, by allosteric modulation, the rate of desensitization of whole cell currents induced by acetylcholine and decreased the maximal amplitude in concentration-dependent manner.

Early postdenervation depolarization develops faster at endplates of hibernating golden hamsters where spontaneous quantal and non-quantal acetylcholine release is very small.

The hyperpolarization produced by the application of curare to the postsynaptic membrane of the diaphragm neuromuscular synapse (H-effect) is a measure of non-quantal release (NQR) of acetylcholine (ACh) from the motor nerve ending. In mouse diaphragm, H-effect was 9.3 mV, significantly lower in awake hamsters (7.1 mV) and very small (1.1 mV) in hibernating hamsters. Also, the initial resting membrane potential (RMP) after dissection was highest in mouse (81.5 mV, inside negative), significantly smaller in awake hamsters (77.9 mV) and lowest in hibernating hamsters (75.1 mV). The early postdenervation depolarization of muscle fiber RMP to about 66-68 mV developed with half-decay time (T1/2) of 120 min in mouse, more rapidly in active hamsters (T1/2=60 min) and even faster in hibernating hamsters (T1/2=25 min) muscles. This reciprocal correlation between the H-effect and the rate of early depolarization indicates that non-quantal release is important for maintaining the resting membrane potential [Vyskocil, F. 2003. Early postdenervation depolarization is controlled by acetylcholine and glutamate via nitric oxide regulation of the chloride transporter. Neurochem. Res. 28, 575-585]. The amplitude of H-effect in mouse and hamster was proportional to the spontaneous quantal release. The frequency of miniature endplate potentials was highest in mouse (1.6 s-1), much smaller in awake hamsters (0.51 s-1) and very small in hibernating hamsters (0.08 s-1). This is in accordance with the idea that non-quantal release depends on the number of vesicles fused with the presynaptic membrane during quantal release [Edwards et al., 1985; Ferguson, S.M., Savchenko, V., Apparsundaram, S., Zwick, M., Wright J., Heilman, C.J., Yi, H., Levey, A.I., Blakely R.D. Vesicular localization and activity-dependent trafficking of presynaptic choline transporters. J. Neurosci. 23 (2003) 9697-9709].

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction.

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 x 10(-4) M), carbachol (CCh, 1 x 10(-5) M) or nicotine (5 x 10(-6) M) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90-120 microm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 x 10(-7) M (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m(o) (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m(o) and dispersed the release (ACh, CCh and nicotine). The effects on m(o) were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.

Dependence of miniature endplate current on kinetic parameters of acetylcholine receptors activation: a model study.

Mathematical modeling was applied to study the dependence of miniature endplate current (MEPC) amplitude and temporal parameters on the values of the rate constants of acetylcholine binding to receptors (k+) when cholinesterase was either active or inactive. The simulation was performed under two different sets of parameters describing acetylcholine receptor activation--one with high and another with low probability (Pohigh and Polow) of receptor transition into the open state after double ligand binding. The dependence of model MEPC amplitudes, rise times, and decay times on k+ differs for set Polow and set Pohigh. The main outcome is that for set Pohigh, the rise time is significantly longer at low values of k+ because of the prolongation of ACh diffusion time to the receptor. For the set Polow, the rise time is shorter at low values of k+, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPC's peak formation.

Early postdenervation depolarization is controlled by acetylcholine and glutamate via nitric oxide regulation of the chloride transporter.

Resting non-quantal acetylcholine (ACh) and probably glutamate (Glu) release from nerve endings activates M1- and NMDA receptor-mediated Ca2+ entry into the sarcoplasm with following activation of NOS and production of NO. This is a trophic message from motoneurons, which keeps the Cl- transport inactive in the innervated sarcolemma. After denervation, the secretion of ACh and Glu at the neuromuscular junction is eliminated within 3-4 h and the production of NO in the sarcoplasm is lowered. As a result, the Cl- influx is probably activated by dephosphorylation of the Cl- transporter with subsequent elevation of intracellular Cl- concentration. The equilibrium Cl- potential becomes more positive and the muscle membrane becomes depolarized.

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction.

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.